Redox- and pH-sensitive glycan (Polysialic acid) derivatives and F127

Subscriber access provided by University of Winnipeg Library

Article

Redox- and pH-sensitive glycan (Polysialic acid) derivatives and F127 mixed micelles for tumor-targeted drug delivery Yihui Deng, Mingqi Liu, Xiang Luo, Qiujun Qiu, Le Kang, Tang Li, Junqiang Ding, Yan Xiong, Zitong Zhao, Chuqing Chang, Jinlei Zan, Xinrong Liu, and Yanzhi Song Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b00687 • Publication Date (Web): 03 Nov 2018 Downloaded from http://pubs.acs.org on November 4, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Molecular Pharmaceutics

1 2 3

Redox- and pH-sensitive glycan (Polysialic acid) derivatives and F127 mixed micelles for tumor-targeted drug delivery

4 5

Mingqi Liu, Xiang Luo, Qiujun Qiu, Le Kang, Tang Li, Junqiang Ding, Yan Xiong,

6

Zitong Zhao, Jinlei Zan, Chuqing, Chang, Xinrong Liu, Yanzhi Song*, Yihui Deng*

7 8

College of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road,

9

Shenyang, Liaoning 110016, China

10 11 12 13 14 15 16 17 18 19 20 21 22 23

Corresponding authors:

24

Yihui Deng, E-mail address: [email protected];

25

Yanzhi Song, E-mail address: [email protected];

26

Telephone: +86 (0) 24 43520553;

27

Fax: +86-24-43520553.

1

ACS Paragon Plus Environment

Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

28

Graphic Abstract

29 30

Abstract

31

With increasing application of PEGylated products, drawbacks are beginning to

32

emerge, such as the ‘PEG dilemma’. Other promising materials may need to be found

33

in the current situation. Endogenous polysialic acid (PSA), which is highly expressed

34

on mammalian, bacterial and malignant surface, may be a promising material in

35

oncology. In this study, a dual-responsive amphiphilic PSA cholesterol derivative

36

(PSA-CS-CH) was synthesized to explore the opportunity of PSA in targeted drug

37

delivery systems. PSA-CS-CH, F127 mixed micelles (PF-M) and pure F127 micelles

38

(F-M) were prepared for comparative anti-tumor experiments. The in vitro experiments

39

showed that modification of PSA-CS-CH significantly increased cytotoxicity and

40

cellular uptake. PF-M had excellent tumor microenvironment response release behavior

41

on acidic media with high GSH levels. The in vivo fluorescence imaging and anti-tumor

42

experiments showed that PF-M had excellent tumor targeting ability and great tumor

43

suppression ability. In summary, biodegradable PSA may contribute to cancer therapy.

44

Keywords: polysialic acid; tumor-targeted; dual-responsive; pH-sensitive; drug

45

delivery system

46

1 Introduction

47

PEGylated nanocarriers confer certain advantages to the drug,, such as prolonged

48

circulatory half- life, increased tumor accumulation, and reduced systemic toxicity1-6.

49

Several PEGylated products have been approved for clinical cancer treatment or clinical

50

trials since 1995. These include PEGylated protein(Adagen®7), PEGylated

51

liposomes(Doxil®8), PEGylated micelles(Genexol®9, NK91110), and PEGylated 2


Page 2 of 38

Page 3 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


52

microemulsions (Aquafol®11). However, PEGylated drugs provide marginal

53

improvement in efficacy over free drugs in clinical settings, especially for solid tumor

54

treatment12-15, such as Doxil®. This is a result of poor intratumoral diffusion13, 16, 17, and

55

unfavorable drug release18-20. In addition, researchers have demonstrated that

56

PEGylation can lead to the ‘PEG dilemma’21,

57

problems associated with PEG have limited its clinical application. There is an urgent

58

need to identify other biodegradable and bioactive substances.

22

and security23-26. Considerable

59

Polysialic acid (PSA) which abundantly presents on the surface of mammalian and

60

bacterial cells27-29 and malignant tumors30-32 is a promising alternative to PEG [25]. PSA

61

(pKa=2.633) is a linear polymer composing of α-2,8 linked sialic acid (SA) monomers.

62

There are numerous reasons for PSA to be potential. First, PSA is an endogenous

63

substance, and could escape from the phagocytosis of immune cells and prolong

64

circulation time32,

65

conjugate was close to 40 hours after intravenous administration to mice. Significant

66

increase in circulation time for IFN-α2b, GCSF, insulin and asparaginase in animal

67

models by polysialylation36. Second, one of the major receptors of SA (selectin) is

68

highly expressed on the surface of tumor cells37,

69

cells39-41, therefore, when the drug carrier circulates to the tumor site, the tumor cells

70

can directly uptake drug through ligand-receptor recognition. PSA could also directly

71

bind to biologically active molecules on the surface of tumor cells through ternary

72

complex formation with FGF2, FGFR, and heparan sulphate (HS)36, 42. Third, PSA also

73

has the advantage of being biodegradable and catabolic products (e.g. SA) which are

74

non-toxic43. Evidence suggests that polysialylation may significantly contribute to

75

cancer therapy.

34, 35.

Gregoriadis et al. demonstrated that the half-life of a PSA

38

and tumor vascular endothelial

76

After intravenous injection, the ideal nanocarriers need to meet requirements of

77

rapid the intracellular release of drugs, long circulation time in the blood and the

78

efficient uptake of tumor cells. Studies have shown that there are such gradients in

79

cancer patients, such as glutathione (GSH) concentration gradients between the cytosol

80

(2-10 mM) and the extracellular (2-20 μM) spaces44, 45 and the pH gradient between

81

lysosomes and endosome (4.5-5.0) and extracellular tumor tissue (6.4-6.8) and normal 3



82

tissue (7.4)46-48. Therefore, a new PSA derivative, PSA-CS-CH (Scheme 1), was

83

sensitive to the pathophysiological characteristics of malignant tissue, and was

84

synthesized to prevent premature drug release while efficiently releasing the drug in

85

target tissue.

86 87

Scheme 1. Synthetic route of (A) CS-CH and (B) PSA-CS-CH.

88

Although PSA-CS-CH has many advantages, the in vitro stability of pure PSA-CS-

89

CH micelles is not high and the particle size is large due to the presence of a large

90

portion of hydrophobic groups in the structure. We can solve this problem with mixed 4


Page 4 of 38

Page 5 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


91

micelles, which can combine the advantages of different nanomaterial to form

92

multifunctional micelles49,

93

biocompatible polymer with moderate cytotoxicity and immunogenicity, which has

94

been approved by FDA as direct and indirect food additives, pharmaceutical ingredients

95

and agricultural products and extensively used in drug delivery systems51-53. Due to its

96

high PEO-PPO ratio, F127 has a low critical micelle concentration (CMC) value, that

97

is, the micelles formed by F127 may be more stable.54. Therefore, a doxorubicin

98

(DOX)-delivering mixed micelle (DOX-PF-M) based on PSA-CS-CH and F127 was

99

designed. The insensitive derivative DOX-F-M, which was composed of F127, but

100

lacked the PSA-CS-CH, was also prepared for comparison. The designed DOX-PF-M

101

was taken up by tumor cells via both receptor-mediated endocytosis and pinocytosis55-

102

59,

103

within the tumor cell, the disulphide bond in CS is sheared off, causing dissociation of

104

DOX-PF-M and drug release. Moreover, once DOX-PF-M is endocytosed, DOX will

105

be released quickly because of the reduced electrostatic forces between PSA and DOX.

106

The in vitro release experiments showed that PF-M can specifically respond to unique

107

GSH and pH microenvironment in tumor cells. The uptake of PF-M by S180 cells was

108

greatly increased and the IC50 was greatly reduced compared to F-M. The results of in

109

vivo imaging and anti-tumor experiments showed that PF-M can accumulate in large

110

numbers in tumor and exerts good cytotoxicity, echoing the in vitro MTT assay results.

111

The experimental flow chart is shown in Scheme 2. The redox and pH dual-responsive

112

micelles offer a powerful platform for the simultaneous enhancement of tumor drug

113

accumulation and rapid and sufficient intracellular drug release to achieve safe and

114

effective cancer therapy.

50.

Pluronic F127, an A-B-A type triblock, non-ionic,

and internalized into tumor cells. Following exposure to the GSH microenvironment

115 116

Scheme 2 Flow chart of in vitro and in vivo targeting and anti-tumor effect verification of PSA-CS5



117

CH modified micelles.

118

2 Experimental

119

2.1 Materials

120

Pluronic F127 (F127, MW =12,600) was supplied by BASF SE (Ludwigshafen,

121

Germany). Poly[2,8-(N-acetylneuraminic acid sodium salt)] (PSA) containing

122

approximately 100 sialic acid (SA) units was isolated from E. coli was provided by

123

Carbosynth China, Ltd. (Shanghai, China). Cystamine (CS) was obtained from TCI

124

Development Co., Ltd. (Shanghai, China). Cholesteryl chloroformate (CHMC) was

125

provided by J&K Scientific Ltd (Shanghai, China). Doxorubicin hydrochloride salt

126

(DOX·HCl) was supplied bym Beijing HuaFeng Co. Ltd. (Beijing, China). N-(3-

127

dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxy-

128

succinimide (NHS) were supplied by China National Medicines Co. Ltd. (Shenyang,

129

China). 3-[4, 5-Dimethyl-thiazol-2-yl]-2, 5-diphenyl tetrazolium bromide (MTT) was

130

supplied by Sigma Aldrich Chemical Co., Ltd. (St. Louis, MO, USA). 1, 1’-

131

Dioctadecyl-3, 3, 3’, 3’-tetramethylindotricarbocyanine iodide (DiR) was acquired

132

from Molecular Probes, Inc. (Eugene, OR, USA). The remaining chemicals were of

133

analytical grade..

134

2.2 Cells and animals

135

S180 cells were purchased from the Chinese Academy of Sciences cell bank

136

(Shanghai, China). Male Kunming mice weighing 18–20 g and Wistar rats weighing

137

180–200 g were obtained from the Experimental Animal Center of Shenyang

138

Pharmaceutical University (Shenyang, China). These animals are free to drink water

139

and eat standard foods. Animal studies were conducted in accordance with the

140

guidelines for animal experiments at Shenyang Pharmaceutical University..

141

2.3 Synthesis of PSA-CS-CH conjugates

6


Page 6 of 38

Page 7 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

142


2.3.1 Synthesis of cystamine-modified CHMC

143

Cystamine-grafted CHMC (CS-CH) was synthesized by coupling CHMC with the

144

primary amino group of CS. Briefly, CS (250 mg, 1 mM) and CHMC (539 mg, 1.2

145

mM) were dissolved in 3 ml formamide (FA) and 1 mL dichloromethane (DCM),

146

respectively, and then mixed. Triethylamine (TEA) (420 μL, 3 mM) was then added to

147

the reaction system and stirred at 25°C under N2 overnight. After completion of the

148

reaction, followed by the extraction of DCM layer which was then removed by rotary

149

evaporation under reduced pressure. The solid was washed with FA, and the resulting

150

FA suspension was dialyzed against distilled water with a 500Da dialysis bag.

151

(Spectrum®, Rancho Dominquez, CA). The retentate obtained was lyophilized and the

152

lyophilisate was washed with FA to remove unreacted CS and lyophilized again. This

153

process was repeated several times until the product weight no longer changes. The

154

final product was characterized by FT-IR (Bruker IFS 55) and MS (Agilent 1100)

155

spectroscopy.

156

2.3.2 Synthesis of amphiphilic PSA-CS-CH conjugates

157

Amphiphilic PSA-CS-CH conjugates were synthesized by the amidation reaction

158

between the carboxylic group of PSA and the primary amino group of CS-CH,

159

catalysed by EDC/NHS. The synthetic route was shown in scheme 1. In brief, PSA (300

160

mg, 1 mM COOH) was dissolved in FA to obtain a polymer solution. EDC (383 mg, 2

161

mM) and sulpho-NHS (115 mg, 1 mM) were added to PSA solution and placed in an

162

ice-water bath for 0.5 h. Next, CS-CH (113 mg, 0.2 mM) dissolved in DCM was added

163

and stirring was continued at 25oC for 4 hours. The ratio between FA and DCM is still

164

3:1. After the reaction was completed, DCM was added to the reaction system to

165

separate FA and DCM, the DCM layer was discarded, and the FA layer was placed in

166

a 1000Da dialysis bag(Spectrum®, Rancho Dominquez, CA) and dialyzed against

167

distilled water.. The retentate was lyophilized and then dissolved in water and filtered

168

to remove insolubles (CS-CH) and lyophilized again. This process was repeated several

169

times until no more insoluble matter appeared. The final product was characterized by

170

FT-IR (Bruker IFS 55) and 1H-NMR (Bruker 600-MHz) spectroscopy.

171

2.3.3 Determination of degree of substitution (DS) of CS-CH on the PSA polymer 7



172

backbone

173

The DS of CS-CH was calculated by ultraviolet-visible spectrophotometry.

174

Excessive dithiothreitol was added to the PSA-CS-CH conjugate to reduce the disulfide

175

bond to free thiol. The disulphide bond content was calculated through the measurement

176

ofthe absorbance of the free sulfhydryl group at 412 nm using the Ellman’s method,

177

and then calculating the sulfhydryl content from the standard curve obtained from the

178

L-cysteine solution (0.05-1.0 mg/mL).

179

2.4 Preparation and characterization of DOX-PF-M and DOX-F-M

180

2.4.1 Preparation of micelles

181

DOX-loaded micelles were prepared by a self-assembly method. Briefly,

182

DOX·HCl was stirred with excess TEA (2 × DOX·HCl) in CHCl3 overnight to obtain

183

a DOX base. F127 (150 mg) and PSA-CS-CH (15 mg) were dissolved in 1 ml of DOX

184

base solution (8 mg DOX base in 1 mL CHCl3), respectively, and stirred for 10 min.

185

The organic solvent was then removed by vacuum evaporation. Distilled water (3 mL)

186

was added dropwise into the mixture of F127, PSA-CS-CH, and DOX base with

187

constant stirring for 20 min. Preparation of F-M was identical to that of PF-M except

188

that PSA-CS-CH is not present. The final PF-M and F-M were obtained by filtering

189

through a 0.22 micron filter to eliminate unencapsulated DOX base and preserved at

190

4°C. DiR-labeled micelles were prepared in the same way as DOX-loaded micelles,

191

except that F127 and PSA-CS-CH were added to the fluorescent probe DiR ethanol

192

solution instead of the DOX base solution. The mean diameter and ζ potential of

193

micelles were measured by a NICOMP380 instrument (NICOMPTM 380, Particle

194

Sizing System, CA, USA).

195

2.4.2 Encapsulation efficiency and loading capacity

196

The encapsulation efficiency and drug loading efficiency of DOX-loaded Micelles

197

were determined by ultraviolet-visible spectrophotometry. Encapsulation efficiency

198

(EE) and drug loading efficiency (DL) were calculated as follows: (1) EE % = (the

199

amount of DOX inside the micelle / the total amount of DOX in the prescription) × 100% 8


Page 8 of 38

Page 9 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


200

and (2) DL % = the amount of DOX inside the micelle / Total amount of all materials

201

in the prescription) × 100%.

202

2.4.3 Morphology

203

A negative staining method was used to stain the micelles and then the morphology

204

was observed with a transmission electron microscope. The micelles were dropped onto

205

a film-coated copper mesh and stained with 2% (w/v) phosphotungstic acid for 1 minute.

206

The samples were measured by using a transmission electron microscope (TEM, JM-

207

1200EX, JEOL Ltd, Japan) after air drying at room temperature.

208

2.5 Critical Micelle Concentration

209

The CMC of the PSA-CS-CH and F127 mixture was determined on a

210

luminescence spectrometer (SpectraMax M3; Molecular Devices LLC, Sunnyvale, CA,

211

USA) by using pyrene as fluorescent probe60, 61. An equivalent volume of 0.1 ml pyrene

212

solution in acetone (1.0×10-5 mol/L) was added into a series of volumetric flasks. The

213

organic solvent was purged under nitrogen. PSA-CS-CH and F127 were weighed and

214

transferred into a 10-mL volumetric flask at a 1:10 ratio. The final concentration of

215

PSA-CS-CH ranged from 1.0 × 10-3 to 1.0 ×10-7 g/mL. The mixture was sonicated for

216

1 h and shaken at a rate of 100 rpm at 25°C overnight. The emission wavelength was

217

fixed at 397 nm, and the excitation wavelength was scanned at wavelengths between

218

300 nm and 350 nm. The CMC of the PSA-CS-CH and F127 mixture was the

219

intersection of the two fitting curve intensity ratios I338 / I334 in the low concentration

220

region and the high concentration region.

221

2.6 In vitro release assay

222

DOX solution (DOX-S), DOX-F-M and DOX-PF-M were placed in a dialysis tube

223

(10 kDa MWCO) and dialyzed against release media of different conditions: pH 7.4

224

PBS with 10 μM GSH (pH 7.4/10 μM GSH), pH 7.4 PBS with 10 mM GSH (pH 7.4/10

225

mM GSH), pH 5.0 PBS with 10 μM GSH (pH 5.0/10 μM GSH), and pH 5.0 PBS with

226

10 mM GSH (pH 5.0/10 mM GSH). The release medium contained 0.1% (w/v) Tween

227

80, which was used to achieve sink conditions. All samples were stirred at 37oC in a

228

water-bath at 100 rpm. At the specified time intervals, the release medium was collected

229

and replaced with an equal volume of fresh PBS. The concentration of DOX was 9



230

measured by a microplate reader (Bio-Rad Laboratories Ltd., Hertfordshire, UK). All

231

samples were measured in parallel three times.

232

2.7 Dilution stability of the micelles

233

With the particle size and encapsulation efficiency used as indicators, the dilution

234

stability of the DOX-loaded micelles was investigated after incubation at 10–80-fold

235

dilution with phosphate buffered saline (pH 7.4) at 37°C for 24 h. The methods for both

236

particle size and encapsulation efficiency measurement are described in Section 2.4.

237

2.8 In vitro cytotoxicity (MTT) studies

238

S180 cells were used to determine the cytotoxicity of different excipients and DOX

239

formulations. Briefly, S180 cells were seeded in 96-well plates at a density of 5000

240

cells per well, then incubated with serial concentrations of excipients and DOX

241

formulations for 48 h. Next, 10 μL of MTT solution (5.0 mg / mL) was added to the

242

wells, and after further incubation for 4 hours, 100 μL of the stop solution(10% SDS/5%

243

isobutanol/0.012 M HCl, w/v/v) was added to dissolve the formazan. The absorbance

244

of the final solution was measured at 570 nm using the microplate reader(Bio-Rad

245

Laboratories Ltd., Hertfordshire, UK). Five replicates of each assay were performed

246

and the IC50 values were calculated by using GraphPad Prism 5.0 software.

247

2.9 In vitro cellular uptake studies

248 249

Flow cytometry and confocal laser scanning microscopy (CLSM) were used to quantitate the uptake behavior of different formulations in S180 cells.

250

CLSM: S180 cells grown as a monolayer were suspended by brief treatment with

251

trypsin and then washed once with fresh culture medium. Aliquots of the S180

252

suspension (1.0 ×105 cells/mL) were incubated with DOX-S, DOX-PF-M, or DOX-F-

253

M (DOX concentration, 5 μg/ml) and diluted in culture medium for 2 h at 37oC. An

254

equal volume of medium was added to the cells of the negative control group. The cells

255

were then washed 3 times with cold PBS and fixed with 4% paraformaldehyde solution

256

followed by 5 μg/ml DAPI staining in PBS and observed using CLSM (Nikon C2

257

Confocal, Tokyo, Japan).

258

Flow cytometry: aliquots of the S180 suspension (1.0 ×105 cells/mL) were

259

incubated with DOX-S, DOX-PF-M, or DOX-F-M (DOX concentration, 5 μg/ml) and 10


Page 10 of 38

Page 11 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


260

diluted in culture medium for 2 h at 37oC. An equal volume of medium was added to

261

the cells of the negative control group. The cells were then washed 3 times with cold

262

PBS and digested with trypsin. The mean fluorescence intensity of the cells was

263

determined by FACS Sort Flow Cytometry (Beckman Coulter, Fullerton, CA, USA).

264

2.10 In vivo circulation studies

265

Wistar rats were randomized into 3 groups of 6 each, and different DOX

266

formulations (DOX dose, 5.0 mg / kg) were administered through the tail vein. At 1, 5,

267

15, and 30 min and 1, 2, 4, 8, 12, and 24 h after injection, heparinized tubes were used

268

to collect blood taken from the orbital sinus of the rats to obtain the anticoagulant

269

sample which were then centrifuged at 1930 g for 10 minutes to obtain plasma

270

(supernatant) and stored in a -20 ° C refrigerator. The pharmacokinetics parameters:

271

half-life (t1/2), area under the drug concentration-time curve values (AUC0-∞), and

272

maximum

273

(Mathematical Pharmacology Professional Committee of China, Shanghai, China).

274

2.11 In vivo fluorescence imaging analysis

concentration

(Cmax)

were

analyzed

using

DAS.2.1.1

software

275

In vivo FX Pro imaging systems (Kodak, Rochester, NY, USA) were used for

276

fluorescence imaging. Mice were injected with DiR-solution (DiR-S), DiR-F-M and

277

DiR-PF-M (DiR dose, 0.6 mg/kg) through the tail vein on day 15 after inoculation of

278

S180 cells in Kunming mice. Fluorescence and X-ray scanning were performed at 1 h,

279

4 h, 8 h and 24 h after intravenous injection using a Kodak in vivo imaging system FX

280

PRO (Bruker, Inc., USA). The experimental excitation emission wavelengths were 720

281

nm and 790 nm, respectively. Mice were anesthetized by inhalation of Gerolan Sol and

282

then moved into the imaging room for scanning. After 24 hours, the mice were

283

sacrificed to dissect the main organs and tumors Fluorescence images were captured

284

using the imaging system and the micelles accumulated in tumors were evaluated by a

285

quantitative region-of-interest (ROI) analysis on the DiR signal variation.

286

2.12 In vivo anti-tumor efficacy 11



287

The ability of DOX preparations to inhibit tumor growth on S180 tumors was

288

evaluated by injecting them into the tail vein of Kunming mice. Briefly, on day 0, 2×106

289

S180 cells were injected subcutaneously into armpit of right front arm of the mice. Then,

290

they were randomly divided into 4 groups of 6 each. DOX-S, DOX-F-M, or DOX-PF-

291

M were administered intravenously on days 3, 6, 9, 12, and 15 after inoculation (DOX

292

dose, 5.0 mg/kg), respectively 62, 63. The control group (5% glucose) was administered

293

equivalent amount of glucose. Body weight and tumor diameter were measured every

294

two days, and the formula for tumor volume was 0.5ab2, where “a” and “b” were the

295

longest diameter and the shortest diameter. Tumor inhibition rate (TIR) includes tumor

296

volume inhibition rate (TIRV, %), calculated from the formula TIRV = (VControl group –

297

VTreatment

298

relationship between body weight, which reflects the quality of life of mice, and tumor

299

volume, we proposed Relative Tumor-inhibition (TI) index as an indicator of anti-

300

tumor pharmacodynamics. Relative TI index was calculated as Relative TI index = TI

301

indexTreated group / TI indexControl group ，TI index = Body weight (g) / Tumor weight (g).

302

The default tumor density was 1g/cm3.

303

2.13 Statistical analysis

group)/VControl group

 100%. Moreover, in order to intuitively reflect the

304

All data are shown as the mean ± standard deviation (SD). Differences between

305

groups were tested for significance using a one-way analysis of variance by the SPSS

306

16 procedure ANOVA. Differences were considered significant at P ≤ 0.05.

307

3. Results and discussion

308

3.1 Synthesis and characterisation of PSA-CS-CH

309

The three reactants were conjugated by the reaction between the carboxyl group

310

of the PSA and the primary amine group of CS and the acyl chloride group of CHMC,

311

as shown in Scheme 1. Due to the varying solubilities of CS and CHMC, a mixed

312

solvent of DCM, FA = 1: 3 (v: v), was selected. During CS-CH purification, DCM was

313

first added to the system and the FA layer was discarded to remove the unreacted excess 12


Page 12 of 38

Page 13 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


314

of CS. DCM was then removed under reduced pressure, the resulting solid was washed

315

with FA and the FA suspension was dialyzed against water to remove the triethylamine

316

salt formed in the reaction. The carboxyl group excess (carboxyl group in PSA: CS-CH

317

= 5: 1, mol:mol) was controlled, and the carboxyl group was activated by incubation

318

with EDC and NHS in an ice bath for 30 min. CS-CH was then added at 25oC and

319

reacted overnight to prevent self-crosslinking of PSA. When the weights of the

320

synthesized products CS-CH and PSA-CS-CH no longer changed, the purification was

321

complete.

322

The structure of CS-CH and PSA-CS-CH were characterised by using MS, 1H

323

NMR, and FT-IR spectroscopy (Fig. 1). The major m/z peaks of CS-CH, appearing at

324

566.5 and 588.3, were assigned to the complexes of [M+H+] and [M+Na+]. PSA is a

325

polymerization of SA in the α-2, 8 manner, therefore, the chemical shift of hydrogen in

326

the C8 position of PSA will become smaller compared to SA. Another characteristic

327

peak of PSA is the methyl in amide structure at the C5 position. After the reaction, the

328

amine group in CS is converted to an amide, so there will be a small increase in the

329

chemical shift of CH2 at the amine group alpha position in CS. There are a large number

330

of alkyl groups in CHMC, and its chemical environment has not changed significantly,

331

so the chemical shift is at the chemical shift of the conventional alkyl group (0.9-1.5

332

ppm). In summary, in the 1H NMR spectrum of PSA-CS-CH (CD3OD, δ ppm), the peak

333

assignment information is as follows: 2.0 (s, CH3-CO-NH-, PSA), 3.17 (q, C8H, PSA),

334

1.1-1.4 (t, -CH2-/-CH3-, CHMC), 3.2 (m, -CH2-, CS). Fig.1C shows the FT-IR spectrum

335

of CS, CHMC, CS-CH, and PSA-CS-CH. Successful synthesis of CS-CH conjugate

336

was demonstrated by the disappearance of acid chloride bonds (1776.5 cm-1) and the

337

appearance of amide (1631.5 cm-1) and disulphide bonds (618.6 cm-1). The amide and

338

disulphide bonds both occurred in the FT-IR spectrum of PSA-CS-CH (1631.6 cm-1

339

and 617.9 cm-1, respectively). The stronger and broader peak appearing around 3400

340

and the attenuation of the alkyl peak intensity appearing around 2900 show the

341

successful coupling of PSA with CS-CH. This is attributed to the large amount of

342

alcoholic hydroxyl groups and carboxyl groups present in PSA. The ratio of alkyl

343

groups in the PSA-CS-CH molecule is lower than that of CS-CH, and thus the relative 13



344

Page 14 of 38

intensity is markedly decreased.

345

The DS defined as the number of CS-CH per 100 carboxyl groups of PSA polymer

346

estimated by the Ellman’s method was 18.9% on a molar basis, indicating that the free

347

amine of cystamine can react with PSA and almost all CS-CH are modified with PSA.

348

349 350

Fig.1 The characterization of CS-CH and PSA-CS-CH. (A) MS spectrum of CS-CH. (B) 1H NMR

351

spectrum of PSA-CS-CH in CD3OD. (C) FT-IR spectrum of CS, CHMC, CS-CH and PSA-CS-CH.

352

3.2 Characterization of DOX-loaded micelles

353

The size of nanocarriers plays a critical role in drug delivery, and can influence 64-67.

354

tumor accumulation, penetration, and treatment

355

below 200 nm can attenuate the uptake of the mononuclear phagocytic system (MPS)

356

and increase accumulation in the tumor due to enhanced permeability and retention

357

(EPR) effects68, 69. In the prescription screening process of micelles, the weight ratio of

358

F127 to PSA-CS-CH was investigated using particle size and polydispersity as

359

indicators. The results are shown in Table S1 and Fig. S1. Finally, we found that when

360

the ratio exceeds 10:1, the particle size and polydispersity of the mixed micelles become

361

sharply large, that is, the micelles formed in a ratio of 10:1 are the most uniform, and

362

the micelles become unstable exceeds this ratio. Therefore, we chose the ratio of 10:1

363

for subsequent experiments. In this study, dynamic light scattering (DLS) 14


Nanocarriers with diameters

Page 15 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


364

measurements revealed that the sizes of DOX-loaded F-M and PF-M were 34.0 ± 1.0

365

nm and 59.1 ± 2.8 nm, respectively. Results of transmission electron microscopy

366

showed that the particle size of the micelles was basically the same as that measured by

367

the DLS method; the micelles existed as spherical nanoparticles, regardless of size

368

(Fig.2 and Fig.S2). Other characteristic parameters were shown in Table 1. The

369

existence of the electric double layer is an important factor for the stability of the

370

dispersion system. When the charged particles get close to each other in the solution,

371

the double electric layers around the particles are cross-linked to generate electrostatic

372

repulsion, thereby stabilizing the preparation. The PSA has an ionizable carboxyl group

373

so the ζ potential of the PF-M increases from -1 mV (F-M) to -14.8 mV, and the PF-M

374

is more stable because of the generation of electric double layer compared to F-M. UV-

375

visible light Spectrophotometry was used to determine the EE and DL of micelles. The

376

drug loading of PF-M is significantly higher than that of F-M (p

Redox- and pH-sensitive glycan (Polysialic acid) derivatives and F127

Recommend Documents